Showing papers by "Shiro Suzuki published in 2013"
TL;DR: Data indicated that OsCOMT1 functioned as a 5-HCAld OMT (OsCAldOMT1) in the biosynthetic pathway to syringyl lignin in rice.
Abstract: Little is known regarding syringyl lignin biosynthesis in rice (Oryza sativa L. cv. Nipponbare). In the present study, the role of rice caffeic acid O-methyltransferase (OsCOMT1, Q6ZD89), was examined. The recombinant OsCOMT1 catalyzed the 5-Omethylation of 5-hydroxyferulate (5-HFA) and 5-hydroxyconiferaldehyde (5-HCAld). 5-HCAld inhibited 5-HFA methylation by this O-methyltransferase (OMT), while 5-HFA mitigated self-inhibition in 5-HCAld methylation. A rice plant in which OsCOMT1 expression was downregulated exhibited weakened cell wall staining with Wiesner reagent in vascular bundle cells and sclerenchyma tissue, compared with wild-type plants. The lignin content of transgenic rice plants was decreased and the syringyl lignin content reduced largely compared with that of the wild type. Taken together, these data indicated that OsCOMT1 functioned as a 5-HCAld OMT (OsCAldOMT1) in the biosynthetic pathway to syringyl lignin.
46 citations
TL;DR: The first fundamental knowledge is provided of E. arundinaceus lignins, a major component of the secondary cell walls of vascular plants, and an obstacle in the conversion of plant cell wall polysaccharides into biofuels.
Abstract: Lignin is a major component of the secondary cell walls of vascular plants, and an obstacle in the conversion of plant cell wall polysaccharides into biofuels. Erianthus spp. are large gramineous plants of interest as potential energy sources. However, lignocelluloses of Erianthus spp. have not been chemically characterized. In this study, we analysed lignins, related compounds, enzymatic saccharification efficiencies, and minerals in the ash of the inner and outer parts of the internode, leaf blade and leaf sheath of Erianthus arundinaceus. Lignins in four organs consisted of guaiacyl, syringyl, and p-hydroxyphenyl units. The ratios of syringyl to guaiacyl lignins and lignin contents ranged from 0.43 to 0.79 and 20 to 28%, respectively, with values highest in the outer part of the internode. The amounts of ferulic acid were similar (7.3–11.8 mg g−1 dry weight of cell-wall material) in all four organs, while there was more p-coumaric acid in the inner part of the internode (44.7 mg g−1 dry weight of cell-wall material) than in other organs (25.7–28.8 mg g−1 dry weight of cell-wall material). The enzymatic saccharification efficiency (24 h reaction time) of the leaf blade was 21.6%, while those of the other organs ranged from 10.0 to 15.2%. The leaf blade had the highest ash content (17.1%); the main inorganic element was silicon. This paper provides the first fundamental knowledge of E. arundinaceus lignins.
41 citations
TL;DR: The results show that deficiency of OsCAD2 causes the bm phenotype in addition to gh, and that the coloration is probably due to the accumulation of cinnamaldehyde-related structures in the lignin.
Abstract: Several brown midrib (bm) mutants have so far been isolated from the C4 grasses, maize, sorghum and pearl millet, but have not been detected in C3 grasses including rice (Oryza sativa). In the present study we characterized the cad2 (cinnamyl alcohol dehydrogenase 2) null mutant isolated from retrotransposon Tos17 insertion lines of Oryza sativa L. ssp. japonica cv. Nipponbare. This mutant exhibited brown-colored midribs in addition to hulls and internodes, clearly indicating both bm and gold hull and internode (gh) phenotypes. The enzymatic saccharification efficiency in the culm of cad2 null mutant was increased by 16.1% than that of the control plants. The lignin content of the cad2 null mutant was 14.6% lower than that of the control plants. Thioacidolysis of the cad2 null mutant indicated the presence of cinnamaldehyde structures in the lignin. Taken together, our results show that deficiency of OsCAD2 causes the bm phenotype in addition to gh, and that the coloration is probably due to the accumulation of cinnamaldehyde-related structures in the lignin. Additionally, this cad2 null mutant was useful to silage purposes and biofuel production.
30 citations
TL;DR: The results indicate that aspen leaves, but not Arabidopsis and tobacco leaves, have a partially active pathway to eugenol that is limited by the level of CFAT activity and thus the flux of this pathway can be increased by the introduction of a single heterologous gene.
29 citations
TL;DR: This is the first report on the isolation and characterization of a thujaplicatin-specific plant OMT, and Quantitative real-time polymerase chain reaction showed that AsTJOMT had the highest expression level in roots compared with other organs.
Abstract: Yatein is an important biosynthetic precursor of the lignan podophyllotoxin. In Anthriscus sylvestris, yatein biosynthesis is preceded by two regioselective methylations involving the intermediates: thujaplicatin and 5-Omethylthujaplicatin. The two methylation steps are most likely catalyzed by plant O-methyltransferases (OMTs). In this paper, we report the isolation and characterization of a cDNA encoding an OMT involved in the first methylation step. The OMT cDNA was isolated from an A. sylvestris cDNA library prepared from roots and young shoots. The OMT was expressed as a recombinant protein using the pET expression system. Of the substrates that were tested, the recombinant OMT exclusively catalyzed regioselective methylation of thujaplicatin to produce 5-O-methylthujaplicatin, and thus was designated as A. sylvestris thujaplicatin OMT (AsTJOMT). Kinetic analysis with its substrate (thujaplicatin) showed that AsTJOMT had a Km value of 3.8 μM and kcat value of 0.29 min−1. Quantitative real-time polymerase chain reaction showed that AsTJOMT had the highest expression level in roots compared with other organs. This was in accordance with plant protein assays in which specific activity for thujaplicatin was significantly higher in roots compared with other organs. To the best of our knowledge, this is the first report on the isolation and characterization of a thujaplicatin-specific plant OMT.
21 citations
TL;DR: Quantitative real-time polymerase chain reaction analysis demonstrated that expression of the CtMROMT gene was synchronized with the O-methyltransferase (OMT) activity profile and arctigenin accumulation in the plant, and demonstrated that CtMRomT is a novel plant OMT for lignan methylation.
Abstract: Lignans are a group of plant phenolic compounds with various biological activities, including antitumor and antioxidant properties. O-Methylation is a critical step in biosynthesis of these compounds. However, little is known about the O-methyltransferase (OMT) enzymes that catalyze lignan O-methylation. We discovered a highly regioselective OMT activity in safflower (Carthamus tinctorius) seeds that catalyzed the methylation of matairesinol, a dibenzylbutyrolactone lignan, into 4′-O-methylmatairesinol (arctigenin) but not 4-O-methylmatairesinol (isoarctigenin). By examining such OMT activity in correlation with OMT transcript abundances during seed development, we cloned a few putative OMT cDNAs and produced their recombinant proteins in Escherichia coli. Among them, one protein exhibited O-methylation activity for matairesinol with the regioselectivity identical to that of the plant protein, and was named C. tinctorius matairesinol OMT (CtMROMT). CtMROMT did not show any detectable OMT activities towards phenylpropanoid monomers under the reaction conditions tested, while it methylated flavonoid apigenin efficiently into 4′-O-methylapigenin (acacetin). However, quantitative real-time polymerase chain reaction analysis demonstrated that expression of the CtMROMT gene was synchronized with the CtMROMT activity profile and arctigenin accumulation in the plant. These results demonstrated that CtMROMT is a novel plant OMT for lignan methylation.
19 citations
TL;DR: The results suggest that ATL54 is an E3 ubiquitin ligase involved in secondary wall biosynthesis and programmed cell death during xylogenesis.
Abstract: Biosynthesis of plant secondary cell walls is controlled by several master transcription factors. Ubiquitin ligases, which mediate ubiquitination of proteins, including transcription factors in the protein degradation pathway, are also believed to regulate secondary wall biosynthesis; however, the exact ubiquitin ligases involved in secondary wall formation have not yet been identified. We conducted a gene co-expression network analysis and found that ATL54, annotated as a RING-finger protein, was highly co-expressed with several transcription factor and enzyme genes involved in secondary wall formation. A recombinant ATL54 protein showed ubiquitin ligase activity. The expression of several biosynthetic genes of cellulose, lignin, and xylan in apical portions of inflorescence stems was up-regulated by ATL54 knock-out. The expression of Xylem Cysteine Peptidase1 (XCP1), which participates in the programmed cell death process of xylem tracheary elements, was down-regulated in middle stem portions of both ATL54-knock-out and ATL54-overexpressed mutants. Alteration of ATL54 expression levels did not, however, affect lignin and polysaccharide content and composition in whole mature stems. Our results suggest that ATL54 is an E3 ubiquitin ligase involved in secondary wall biosynthesis and programmed cell death during xylogenesis.
14 citations
TL;DR: The results indicate that ATL54 expression is regulated by MYB46, and support the view that ATL 54 has a role in secondary wall formation.
Abstract: We previously characterized Arabidopsis Tóxicos en Levadura54 (ATL54), a ubiquitin ligase associated with secondary cell wall formation. The ATL54 gene is co-expressed with secondary wall-associated genes, and the knock-out of ATL54 up-regulates the expression of cellulose, lignin, and xylan biosynthetic genes in apical stem portions of four-week-old plants. Here, we report the tissue-level localization patterns and the regulation of ATL54 expression. The β-glucuronidase (GUS) reporter gene driven by the ATL54 promoter was significantly expressed in interfascicular fibers, xylary fibers, and vessels in inflorescence stems. A transient transfection assay using Arabidopsis T87 cells showed that the expression of the firefly luciferase gene driven by the ATL54 promoter was activated by MYB46, which is a key transcriptional activator of secondary wall formation. In addition, the electrophoretic mobility of ATL54 promoter fragments was shifted by a recombinant MYB46 protein. These results indicate that ATL54 expression is regulated by MYB46, and support the view that ATL54 has a role in secondary wall formation.
7 citations
31 Oct 2013
TL;DR: The gene expression results support the proposed idea that FpOAH plays more significant role than FpGLOXDH in oxalate biosynthesis in F. palustris.
Abstract: The brown-rot fungus Fomitopsis palustris possess two oxalate-producing enzymes: oxaloacetate acetylhydrolase (Fomitopsis palustris oxaloacetate acetylhydrolase, FpOAH), which catalyzes hydrolysis of oxaloacetate, and cytochrome c dependent glyoxylate dehydrogenase (Fomitopsis palustris glyoxylate dehydrogenase, FpGLOXDH), which catalyzes dehydrogenation of glyoxylate. Oxaloacetate was regarded as the predominant precursor for oxalate, because greater FpOAH activity was detected than FpGLOXDH activity. In this study, a 1080-bp cDNA encoding FpOAH was cloned. Recombinant FpOAH showed oxaloacetate acetylhydrolase (OAH, EC 3.7.1.1) activity, which confirmed that the isolated cDNA encoded FpOAH. Expression of the gene encoding FpOAH was 22.0-140.8 times greater than that encoding FpGLOXDH, depending on culture times. The gene expression results support our proposed idea that FpOAH plays more significant role than FpGLOXDH in oxalate biosynthesis in F. palustris.
4 citations
TL;DR: Three cDNAs encoding cinnamyl alcohol dehydrogenase (CAD) from Carthamus tinctorius are isolation and characterization and the product yields in plant protein assays were strongly correlated with gene expressions of CtCAD2 andCtCAD3 in the respective organs.
Abstract: We report the isolation and characterization of three cDNAs encoding cinnamyl alcohol dehydrogenase (CAD) from Carthamus tinctorius (safflower). All three recombinant CADs were able to reduce coniferaldehyde and sinapaldehyde into coniferyl alcohol and sinapyl alcohol, respectively, and were designated as CtCAD1, CtCAD2, and CtCAD3. Phylogenetic analysis of CAD amino acid sequences and homology modeling revealed that CtCAD1 and CtCAD3 were closely related to the sinapaldehyde-specific aspen (Populus tremuloides) sinapyl alcohol dehydrogenase (PtreSAD). CtCAD2 was in a clade containing class I plant CADs. Gas chromatography-mass spectrometry-based kinetic analysis using two different substrates, coniferaldehyde and sinapaldehyde, indicated that the CtCADs showed no strong preference for either substrate. CtCAD2 has the highest catalytic efficiency (kcat/Km) (81.49 mM−1 min−1 and 95.3 mM−1 min−1 for coniferaldehyde and sinapaldehyde, respectively) compared with the other CtCADs. Inhibition kinetics showed that coniferaldehyde was a stronger inhibitor than sinapaldehyde for all CtCADs. Quantitative real-time PCR revealed that CtCAD2 was expressed at higher levels than CtCAD1 and CtCAD3 in all samples, except developing seeds at 3 days after flowering, where CtCAD1 had a higher expression level. In plant protein assays with coniferaldehyde and sinapaldehyde, plant protein extracted from seeds at 7 days after flowering, showed the highest specific activity. The product yields in plant protein assays were strongly correlated with gene expressions of CtCAD2 and CtCAD3 in the respective organs.
3 citations
31 Oct 2013
TL;DR: Characterization of lignocellulose in Erianthus arundinaceus and identification of the genes that encode antitumor lignan biosynthetic enzymes are identified.
Abstract: Characterization of lignocellulose in Erianthus arundinaceus and identification of the genes that encode antitumor lignan biosynthetic enzymes Author(s) Yamamura, Masaomi; Ragamustari, Safendrri Komara; Suzuki, Shiro; Umezawa, Toshiaki Citation Sustainable humanosphere : bulletin of Research Institute for Sustainable Humanosphere Kyoto University (2013), 9: 3-3 Issue Date 2013-10-31 URL http://hdl.handle.net/2433/185677 Right Type Departmental Bulletin Paper Textversion publisher
01 Feb 2013
TL;DR: Development of high-throughput characterization of lignocelluloses and the analysis of lIGNo celluloses from Jatropha curcas is developed.
Abstract: Development of high-throughput characterization of lignocelluloses and the analysis of lignocelluloses from Jatropha curcas Author(s) Umezawa, Toshiaki; Suzuki, Shiro Citation Sustainable humanosphere : bulletin of Research Institute for Sustainable Humanosphere Kyoto University (2013), 8: 3-3 Issue Date 2013-02-01 URL http://hdl.handle.net/2433/182615 Right Type Departmental Bulletin Paper Textversion publisher